Enhancement of fibronectin fibrillogenesis and bone formation by basic fibroblast growth factor via protein kinase C-dependent pathway in rat osteoblasts

Chih Hsin Tang, Rong Sen Yang, Tsang-Hai Huang, Shing Hwa Liu, Wen Mei Fu

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

Fibronectin (Fn) is involved in early stages of bone formation and basic fibroblast growth factor (bFGF) is an important factor regulating osteogenesis. We here found that bFGF enhanced extracellular assembly from either endogenously released or exogenously applied soluble Fn in primary cultured osteoblasts. bFGF increased protein levels of Fn using Western blotting analysis. Protein kinase C (PKC) inhibitors such as H7, 3-[1-[3-(amidinothio)propyl-1H-indol-3-yl] -3-(1-methyl-1H-indol-3-yl)maleimide (Bisindolylmaleimide IX), methanesulfonate (Ro 318220,) or 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H- indolo[2,3-a]pyrrolo[3,4-c] carbazole (Gö 6976) antagonized the increase of Fn protein by bFGF. Treatment of osteoblasts with bFGF increased membrane translocation of various isoforms of PKC, including α, β, ε, and δ. However, treatment with antisense of various PKC isoforms demonstrated that α and β isozymes play important roles in the enhancement action of bFGF on Fn assembly. Down-regulation of PKC by prolonged treatment with 1 μM 12-O-tetradecanoylphorbol-13 acetate for 24 h inhibited the potentiating action of bFGF. It has been reported that α5β1 integrin is related to Fn fibrillogenesis, and immunocytochemistry showed that bFGF treatment increased the clustering of α5 integrins. Flow cytometry analysis demonstrated that bFGF increased cell surface expression of α5 and β1 integrins and PKC inhibitors antagonized the increase by bFGF. Local administration of bFGF into the metaphysis of the tibia via the implantation of a needle cannula significantly increased the protein levels of Fn in the area of trabecular spongiosa, which was inhibited by coadministration of PKC inhibitors. Furthermore, local injection of bFGF increased the bone volume of secondary spongiosa in tibia, which was significantly antagonized by PKC inhibitors. These results suggest that bFGF increased bone formation and Fn fibrillogenesis both in vitro and in vivo via PKC-dependent pathway.

Original languageEnglish
Pages (from-to)440-449
Number of pages10
JournalMolecular Pharmacology
Volume66
Issue number3
Publication statusPublished - 2004 Sep 1

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Fibroblast Growth Factor 2
Osteoblasts
Fibronectins
Osteogenesis
Protein Kinase C
Protein C Inhibitor
Protein Kinase Inhibitors
Integrins
Tibia
Protein Isoforms
Proteins
Tetradecanoylphorbol Acetate
Therapeutics
Isoenzymes
Needles
Cluster Analysis
Flow Cytometry
Down-Regulation
Western Blotting
Immunohistochemistry

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Pharmacology

Cite this

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title = "Enhancement of fibronectin fibrillogenesis and bone formation by basic fibroblast growth factor via protein kinase C-dependent pathway in rat osteoblasts",
abstract = "Fibronectin (Fn) is involved in early stages of bone formation and basic fibroblast growth factor (bFGF) is an important factor regulating osteogenesis. We here found that bFGF enhanced extracellular assembly from either endogenously released or exogenously applied soluble Fn in primary cultured osteoblasts. bFGF increased protein levels of Fn using Western blotting analysis. Protein kinase C (PKC) inhibitors such as H7, 3-[1-[3-(amidinothio)propyl-1H-indol-3-yl] -3-(1-methyl-1H-indol-3-yl)maleimide (Bisindolylmaleimide IX), methanesulfonate (Ro 318220,) or 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H- indolo[2,3-a]pyrrolo[3,4-c] carbazole (G{\"o} 6976) antagonized the increase of Fn protein by bFGF. Treatment of osteoblasts with bFGF increased membrane translocation of various isoforms of PKC, including α, β, ε, and δ. However, treatment with antisense of various PKC isoforms demonstrated that α and β isozymes play important roles in the enhancement action of bFGF on Fn assembly. Down-regulation of PKC by prolonged treatment with 1 μM 12-O-tetradecanoylphorbol-13 acetate for 24 h inhibited the potentiating action of bFGF. It has been reported that α5β1 integrin is related to Fn fibrillogenesis, and immunocytochemistry showed that bFGF treatment increased the clustering of α5 integrins. Flow cytometry analysis demonstrated that bFGF increased cell surface expression of α5 and β1 integrins and PKC inhibitors antagonized the increase by bFGF. Local administration of bFGF into the metaphysis of the tibia via the implantation of a needle cannula significantly increased the protein levels of Fn in the area of trabecular spongiosa, which was inhibited by coadministration of PKC inhibitors. Furthermore, local injection of bFGF increased the bone volume of secondary spongiosa in tibia, which was significantly antagonized by PKC inhibitors. These results suggest that bFGF increased bone formation and Fn fibrillogenesis both in vitro and in vivo via PKC-dependent pathway.",
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Enhancement of fibronectin fibrillogenesis and bone formation by basic fibroblast growth factor via protein kinase C-dependent pathway in rat osteoblasts. / Tang, Chih Hsin; Yang, Rong Sen; Huang, Tsang-Hai; Liu, Shing Hwa; Fu, Wen Mei.

In: Molecular Pharmacology, Vol. 66, No. 3, 01.09.2004, p. 440-449.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Enhancement of fibronectin fibrillogenesis and bone formation by basic fibroblast growth factor via protein kinase C-dependent pathway in rat osteoblasts

AU - Tang, Chih Hsin

AU - Yang, Rong Sen

AU - Huang, Tsang-Hai

AU - Liu, Shing Hwa

AU - Fu, Wen Mei

PY - 2004/9/1

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N2 - Fibronectin (Fn) is involved in early stages of bone formation and basic fibroblast growth factor (bFGF) is an important factor regulating osteogenesis. We here found that bFGF enhanced extracellular assembly from either endogenously released or exogenously applied soluble Fn in primary cultured osteoblasts. bFGF increased protein levels of Fn using Western blotting analysis. Protein kinase C (PKC) inhibitors such as H7, 3-[1-[3-(amidinothio)propyl-1H-indol-3-yl] -3-(1-methyl-1H-indol-3-yl)maleimide (Bisindolylmaleimide IX), methanesulfonate (Ro 318220,) or 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H- indolo[2,3-a]pyrrolo[3,4-c] carbazole (Gö 6976) antagonized the increase of Fn protein by bFGF. Treatment of osteoblasts with bFGF increased membrane translocation of various isoforms of PKC, including α, β, ε, and δ. However, treatment with antisense of various PKC isoforms demonstrated that α and β isozymes play important roles in the enhancement action of bFGF on Fn assembly. Down-regulation of PKC by prolonged treatment with 1 μM 12-O-tetradecanoylphorbol-13 acetate for 24 h inhibited the potentiating action of bFGF. It has been reported that α5β1 integrin is related to Fn fibrillogenesis, and immunocytochemistry showed that bFGF treatment increased the clustering of α5 integrins. Flow cytometry analysis demonstrated that bFGF increased cell surface expression of α5 and β1 integrins and PKC inhibitors antagonized the increase by bFGF. Local administration of bFGF into the metaphysis of the tibia via the implantation of a needle cannula significantly increased the protein levels of Fn in the area of trabecular spongiosa, which was inhibited by coadministration of PKC inhibitors. Furthermore, local injection of bFGF increased the bone volume of secondary spongiosa in tibia, which was significantly antagonized by PKC inhibitors. These results suggest that bFGF increased bone formation and Fn fibrillogenesis both in vitro and in vivo via PKC-dependent pathway.

AB - Fibronectin (Fn) is involved in early stages of bone formation and basic fibroblast growth factor (bFGF) is an important factor regulating osteogenesis. We here found that bFGF enhanced extracellular assembly from either endogenously released or exogenously applied soluble Fn in primary cultured osteoblasts. bFGF increased protein levels of Fn using Western blotting analysis. Protein kinase C (PKC) inhibitors such as H7, 3-[1-[3-(amidinothio)propyl-1H-indol-3-yl] -3-(1-methyl-1H-indol-3-yl)maleimide (Bisindolylmaleimide IX), methanesulfonate (Ro 318220,) or 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H- indolo[2,3-a]pyrrolo[3,4-c] carbazole (Gö 6976) antagonized the increase of Fn protein by bFGF. Treatment of osteoblasts with bFGF increased membrane translocation of various isoforms of PKC, including α, β, ε, and δ. However, treatment with antisense of various PKC isoforms demonstrated that α and β isozymes play important roles in the enhancement action of bFGF on Fn assembly. Down-regulation of PKC by prolonged treatment with 1 μM 12-O-tetradecanoylphorbol-13 acetate for 24 h inhibited the potentiating action of bFGF. It has been reported that α5β1 integrin is related to Fn fibrillogenesis, and immunocytochemistry showed that bFGF treatment increased the clustering of α5 integrins. Flow cytometry analysis demonstrated that bFGF increased cell surface expression of α5 and β1 integrins and PKC inhibitors antagonized the increase by bFGF. Local administration of bFGF into the metaphysis of the tibia via the implantation of a needle cannula significantly increased the protein levels of Fn in the area of trabecular spongiosa, which was inhibited by coadministration of PKC inhibitors. Furthermore, local injection of bFGF increased the bone volume of secondary spongiosa in tibia, which was significantly antagonized by PKC inhibitors. These results suggest that bFGF increased bone formation and Fn fibrillogenesis both in vitro and in vivo via PKC-dependent pathway.

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